Catechin inhibits ox-LDL-induced ferroptosis in vascular smooth muscle cells to alleviate and stabilize atherosclerosis

Abstract

Atherosclerosis (AS) is a chronic, progressive vascular disease marked by lipid deposition in the arterial intima, vascular wall thickening, luminal narrowing, and compromised blood flow. Although macrophage-derived foam cells are well-studied, vascular smooth muscle cells (VSMCs) also substantially contribute to AS, particularly when they transition into foam cells under oxidative stress. Accumulating evidence suggests that ferroptosis-an iron-dependent, regulated cell death mechanism characterized by lipid peroxidation-exacerbates AS pathology through oxidative damage and vascular dysfunction. Catechin, a potent antioxidant abundant in green tea, has demonstrated efficacy in reducing oxidative stress; however, its role in suppressing VSMC ferroptosis induced by oxidized low-density lipoprotein (ox-LDL) remains unclear. Here, we evaluated catechin's capacity to protect VSMCs against ox-LDL-induced ferroptosis, focusing on its modulation of the Nrf2/SLC7A11/GPX4 axis. Mouse vascular smooth muscle (MOVAS) cells were incubated with ox-LDL to induce foam cell formation and ferroptosis. We assessed intracellular iron, lipid peroxidation, reactive oxygen species (ROS), and antioxidant defenses and examined mitochondrial ultrastructure via transmission electron microscopy (TEM). Ferroptosis-related proteins were measured by Western blot, immunofluorescence, and qPCR. In vivo, ApoE^-/- mice on a high-fat diet (HFD) underwent partial carotid ligation with local catechin administration to investigate plaque formation and ferroptosis in arterial tissue. Our results show that catechin reduced intracellular Fe²⁺, decreased ROS and malondialdehyde (MDA) levels, and preserved mitochondrial integrity in ox-LDL-exposed MOVAS cells. Catechin also enhanced GSH and SOD levels and restored GPX4, SLC7A11, and Nrf2 expression, thereby reducing foam cell formation. In ApoE-/- mice, catechin reduced plaque size, mitigated lipid deposition, and upregulated GPX4, SLC7A11, and Nrf2 in the arterial wall. Collectively, these findings confirm that catechin prevents ox-LDL-induced ferroptosis in VSMCs by activating the Nrf2/SLC7A11/GPX4 pathway, highlighting its potential therapeutic value for atherosclerosis. This study provides additional evidence for the role of dietary polyphenols in regulating ferroptosis within VSMCs.

Keywords: GPX4; Nrf2 pathway; atherosclerosis; catechin; ferroptosis; ox-LDL; oxidative stress; vascular smooth muscle cells.

Figure 1

Catechin's effect on MOVAS viability, optimal ox-LDL dose, and foam cell formation. (A) A CCK-8 assay shows that catechin at concentrations >200 μM significantly reduces MOVAS viability at 36–48 h. (B–D) Increasing ox-LDL doses (0–100 μg/mL) increase total cholesterol (TC), free cholesterol (FC) levels, as well as the cholesteryl ester to total cholesterol (CE/TC) ratio. At ox-LDL concentrations ≥80 μg/mL, ox-LDL induces an esterification rate (CE/TC), and the CE/TC ratio exceeds 50%, indicating foam cell formation. (E–G) Co-incubation with catechin (100 or 200 μM) lowers total and free cholesterol levels, reducing the esterification CE/TC ratio to below 50%. (H, I) Oil Red O staining shows the extensive lipid droplets in the ox-LDL group, and this accumulation is reduced by catechin treatment. Data are mean ± SD (n = 5). ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2

Catechin mitigates iron- and reactive oxygen species (ROS)-dependent oxidative stress in MOVAS cells. (A) FerroOrange staining reveals elevated Fe²⁺ in cells treated with 100 μg/mL ox-LDL, which is reduced by catechin in a dose-dependent manner. (B) DCFH-DA flow cytometry indicates that catechin treatment decreases oxLDL-induced ROS levels. (C) MDA levels, a marker of lipid peroxidation, are increased by oxLDL and lowered by catechin. (D, E) Catechin treatment restores SOD and GSH levels depleted by ox-LDL. (F) Transmission electron micrographs show mitochondrial condensation with collapsed cristae in the ox-LDL group. These changes are normalized by catechin. Data are mean ± SD (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

Catechin rescues ferroptosis-related protein expression in MOVAS. Western blot and quantification show that ox-LDL downregulates GPX4 (A), SLC7A11 (B), and Nrf2 (C). Catechin substantially reverses this effect, with the effect on Nrf2 being most pronounced at 200 μM. Data are mean ± SD (n = 5). ns, not significant; *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 4

Catechin reduces atherosclerotic lesion formation in ApoE^−/− mice subjected to partial carotid ligation. (A) Schematic of partial ligation and local catechin delivery, as detailed in the Methods. (B) Ultrasound imaging reveals diminished blood flow and a reduced arterial lumen diameter in the ligated LCA. (C, D) Representative micrographs showing plaques in carotid arteries (C) and hematoxylin and eosin (H&E) staining of tissue sections (D) and Oil Red O staining (E, F) en face and of cross sections of ligated arteries demonstrate smaller plaques and reduced lipid accumulation in catechin-treated (CAT) mice compared to mock-treated (MOD) mice. (G) Shows quantification of lesion burden and lipid content in the ligated arteries. (H) Serum lipid profiles, including total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c), are unchanged. Data are mean ± SD (n = 5). ns, not significant; **p < 0.01.

Figure 5

Catechin upregulates ferroptosis-related proteins in arteries. (A–C) Immunofluorescence of arterial sections shows alpha-smooth muscle actin (α-SMA, red) co-expression with GPX4 (A), SLC7A11 (B), or Nrf2 (C) (green) in both mock (Mod) and catechin-treated mice, with stronger immunoreactivities for these markers in catechin-treated arteries, consistent with reduced ferroptotic stress and more stable lesions. (D–F) Graphs depicting smooth muscle expression of GPX4, SLC7A11, and Nrf2, presented as the ratio of each marker's fluorescent signal to that of α-SMA. Data are mean ± SD (n = 5). *p < 0.05, **p < 0.01.